Mass spectrometry assay for plasma-renin

ABSTRACT

Provided are methods for measuring renin activity in a plasma sample using mass spectrometry. The methods generally involve ionizing purified angiotensin 1 from the sample and detecting the amount of angiotensin 1 ions generated. The amount of detected angiotensin 1 ions are then related to the amount of angiotensin 1 generated in the sample, which in turn is related to renin activity in the sample.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to PCT Application Serial NumberPCT/US2009/0531899 filed Aug. 7, 2009, and U.S. application Ser. No.12/189,092 filed Aug. 8, 2008, both of which are incorporated herein byreference in their entirety including all figures and tables.

FIELD OF THE INVENTION

The invention relates to the measurement of renin activity. In aparticular aspect, the invention relates to methods for measurement ofplasma renin activity by HPLC-mass spectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

As discussed in Fredline et al. Clin. Chem. 45: 659-664 (1999), renin isa proteolytic enzyme secreted into blood by the juxtaglomerular cells ofthe kidney. Renin acts on angiotensinogen, to produce a decapeptidereferred to as angiotensin 1 (Ang1). Ang1 is further cleaved byangiotensin-converting enzyme to form an octapeptide, referred to asangiotensin 2 (Ang2). Ang2 stimulates cell growth, renal tubuletransport of sodium, and aldosterone release. Ang2 is one of the mostpotent vasopressors in humans and plays an important role in bloodpressure regulation. Direct measurement of Ang2 is difficult because ofits very low circulating concentrations and extremely short half-life:Ang1, which is more stable than Ang2, provides a better analyte tomeasure the state of the renin-angiotensin system. Determination of a“plasma renin activity” (PRA) from the rate of generation of Ang1 isused clinically for the diagnosis and management of hypertension.

The classical method for the determining PRA is radioimmunoassay (RIA),see Sealey, Clin. Chem. 37:1811-1819 (1991); Shionoiri et al., Horm Res.37:171-175 (1992). A typical radioimmunoassay is performed by thesimultaneous preparation of a series of standard and unknown mixtures intest tubes, each containing identical concentrations of labeled antigenand specific antibody. After an appropriate reaction time, theantibody-bound (B) and free (F) fractions of the labeled antigen areseparated by one of a variety of techniques. The B/F ratios in thestandards are plotted as a function of the concentration of unlabeledantigen (standard curve), and the unknown concentration of antigen isdetermined by comparing the observed B/F ratio with the standard curve.Radioimmunoassay methods are based on competitive binding principles,and the antibodies used can undergo nonspecific binding with otherplasma proteins such as endogenous angiotensins. This potentialcross-reactivity can cause overestimation of the PRA. Another approachis to use HPLC to isolate Ang1 from other angiotensins beforequantification with RIA (see examples from Meng et al., J. Am. Soc.Nephrol. 6: 1209-1215 (1995); Meng et al., J. Chromatogr. 21, 614(1):19-25 (1993); Kohara et al., Peptides 12: 1135-1141 (1991)). These HPLCmethods for the quantification of Ang1 have been developed usingultraviolet, fluorescence and mass spectrometer detection (see examplesfrom Klickstein et al. Anal Biochem 120: 146-150 (1982); Miyazaki et al.J. Chromatogr. 490: 43-51 (1989) and Fredline et al. Clin. Chem. 45:659-664 (1999)).

For example, Fredline et al. describes measurement of plasma reninactivity with the use of HPLC-electrospray-tandem mass spectrometry. Indoing this measurement, Fredline et al. incubates plasma samples in thepresence of a water-sensitive enzyme inhibitor (i.e.,phenylmethylsulphonyl fluoride (PMSF)) and measures the amount ofangiotensin 1 generated during incubation by observing the collisioninduced dissociation of precursor ions with a (m/z) of 649.

SUMMARY OF THE INVENTION

The present invention provides methods for measuring the amount ofangiotensin 1 and for measuring plasma renin activity in a sample bymass spectrometry, including tandem mass spectrometry.

In one aspect, methods are provided for measuring the amount ofangiotensin 1 in a sample. The methods may include: (a) ionizingangiotensin 1 in the sample to produce one or more ions detectable bymass spectrometry; (b) detecting the amount of angiotensin 1 ion(s) bymass spectrometry, wherein the ions are selected from the groupconsisting of ions with a mass/charge ratio of 433.0±0.5, 619.4±0.5,647.4±0.5 and 1297±0.5; and (c) using the amount of angiotensin 1 ion(s)detected to measure the amount of angiotensin 1 in the sample. In someembodiments, the limit of quantitation of the methods is less than orequal to 0.1 ng/mL; such as less than or equal to 0.05 ng/mL; such asabout 0.03 ng/mL. In further embodiments, the method comprises purifyingangiotensin 1 from the sample by high performance liquid chromatography(HPLC). In some embodiments, the methods further comprise purifyingangiotensin 1 in the sample with a solid-phase extraction column. Inother embodiments, the amount of the angiotensin 1 ion(s) is related toamount of angiotensin 1 in the sample by comparison to an internalstandard. In some embodiments, a degradation standard may be used todetermine the degree of degradation of angiotensin 1 during a reninactivity assay.

In another aspect, methods are provided for measuring the amount ofangiotensin 1 generated by renin in a sample. The methods may include:(a) ionizing angiotensin 1 from the sample to produce one or more ions;and (b) detecting the amount of at least one said ion(s) by massspectrometry wherein said ion is selected from the group consisting ofions with a mass/charge ratio of 433.0±0.5, 619.4±0.5, 647.4±0.5 and1297±0.5; and wherein the amount of angiotensin 1 ion(s) detectedprovides a measure of the amount of angiotensin 1 in the sample. In someembodiments, a degradation standard may be used to determine the degreeof degradation of angiotensin 1 during a renin activity assay.

In another aspect, methods are provided for measuring the amount ofangiotensin 1 in a sample. The methods may include: (a) incubating thesample premixed with a water stable protease inhibitor that is noteffective against renin under conditions suitable for the generation ofangiotensin 1 by renin in the sample; (b) purifying angiotensin 1 fromthe sample by liquid chromatography; (c) ionizing purified angiotensin 1to produce one or more ions detectable by mass spectrometry; (d)detecting the amount of one or more angiotensin ion(s) by massspectrometry, and (e) using the amount of ion(s) detected to measure theamount of angiotensin 1 in the sample. In some embodiments, the limit ofquantitation of the method is less than or equal to 0.1 ng/mL; less thanor equal to 0.05 ng/mL; and about 0.03 ng/mL. In further embodiments,the ions generated by mass spectrometry are selected from the groupconsisting of ions with a mass/charge ratio of 433.0±0.5, 619.4±0.5,647.4±0.5 and 1297±0.5. In related embodiments, the ions comprise aprecursor ion with a mass/charge ratio of 433.0±0.5, and one or morefragment ions selected from the group consisting of ions with amass/charge ratio of 619.4±0.5 and 647.4±0.5. In other embodiments, theamount of angiotensin 1 ion(s) is related to the amount of angiotensin 1in the test sample by comparison to an internal standard. In otherembodiments, the methods further comprise purifying angiotensin 1 in thesample with a solid-phase extraction column. In other embodiments, thewater stable protease inhibitor is aminoethylbenzylsulfonyl fluoride. Insome embodiments, a degradation standard may be used to determine thedegree of degradation of angiotensin 1 during a renin activity assay.

In another aspect, methods are provided for measuring the amount ofangiotensin 1 in a sample. The methods may include: (a) incubating thesample under conditions suitable for the generation of angiotensin 1 byrenin in the sample; (b) purifying angiotensin 1 from said sample bysolid phase extraction; (c) further purifying angiotensin 1 followingstep (b) by liquid chromatography with on-line processing; (d) ionizingpurified angiotensin 1 from step (c) to produce one or more ionsdetectable by mass spectrometry; and (e) detecting the amount of one ormore angiotensin 1 ion(s) by mass spectrometry, (f) using the amount ofion(s) detected to measure the amount of angiotensin 1 in the sample. Insome embodiments, the liquid chromatography is high performance liquidchromatography (HPLC). In other embodiments, the limit of quantitationof the methods is less than or equal to 0.1 ng/mL; less than or equal to0.05 ng/mL; or about 0.03 ng/mL. In further embodiments, the methodsinclude generating ions comprising one or more ions selected from thegroup consisting of ions with a mass/charge ratio of 433.0±0.5,619.4±0.5, 647.4±0.5 and 1297±0.5. In related embodiments, the methodsinclude generating precursor ions of angiotensin 1 in which at least oneof the precursor ions has a mass/charge ratio of 433.0±0.5. In relatedembodiments, the methods may include generating one or more fragmentions of an angiotensin 1 precursor ion in which at least one of thefragment ions has a mass/charge ratio of 619.4±0.5 or 647.4±0.5. Infurther embodiments, the incubation occurs in the presence of a waterstable protease inhibitor that is not effective against renin. Incertain preferred embodiments, the water stable protease inhibitor isaminoethylbenzylsulfonyl fluoride. In some embodiments, the amount ofangiotensin 1 ion(s) is related to the amount of angiotensin 1 in thetest sample by comparison to an internal standard. In some embodiments,a degradation standard may be used to determine the degree ofdegradation of angiotensin 1 during a renin activity assay.

In another aspect, methods are provided for measuring renin activity ina sample. The methods may include: (a) incubating the sample underconditions suitable for the generation of angiotensin 1 by renin in thesample; (b) purifying angiotensin 1 from the sample by solid phaseextraction; (c) further purifying angiotensin 1 by liquid chromatographywith on-line processing; (d) ionizing purified angiotensin 1 to produceone or more ions detectable by mass spectrometry; (e) detecting theamount of one or more angiotensin 1 ion(s) by mass spectrometry; (f)using the amount of ion(s) detected to the amount of angiotensin 1 inthe sample; and (g) using the quantity of angiotensin 1 in the sample tocalculate renin activity in the sample. In some embodiments, the liquidchromatography is high performance liquid chromatography (HPLC). Inanother embodiments, the limit of quantitation of the methods is lessthan or equal to 0.1 ng/mL; less than or equal to 0.05 ng/mL; or about0.03 ng/mL. In further embodiments, the methods include generatingprecursor ions of angiotensin 1 in which at least one of the precursorions has a mass/charge ratio of 433.0±0.5, and generating one or morefragment ions of an angiotensin 1 precursor ion selected from the groupconsisting of ions with a mass/charge ratio of 619.4±0.5 or 647.4±0.5.In some embodiments, the incubation occurs in the presence of a waterstable protease inhibitor. In related embodiments, the water stableprotease inhibitor is aminoethylbenzylsulfonyl fluoride. In someembodiments, the amount of the angiotensin 1 ion(s) is related to theamount of angiotensin 1 in the test sample by comparison to an internalstandard. In some embodiments, a degradation standard may be used todetermine the degree of degradation of angiotensin 1 during a reninactivity assay.

In another aspect, methods are provided for measuring renin activity ina sample. The methods include: (a) incubating the sample premixed with awater stable protease inhibitor that is not effective against reninunder conditions suitable for the generation of angiotensin 1 by reninin the sample; (b) purifying angiotensin 1 by liquid chromatography; (c)ionizing the purified angiotensin 1 to produce one or more ionsdetectable by mass spectrometry; (d) detecting the amount of one or moreangiotensin 1 ion(s) by mass spectrometry; (e) using the amount ofion(s) detected to measure the amount of angiotensin 1 in said sample;and (f) using the amount of angiotensin 1 in the sample to calculaterenin activity in the sample. In some preferred embodiments, the liquidchromatography is high performance liquid chromatography (HPLC). Inanother embodiments, the limit of quantitation of the methods is lessthan or equal to 0.1 ng/mL; less than or equal to 0.05 ng/mL; or about0.03 ng/mL. In further embodiments, the methods include generatingprecursor ions of angiotensin 1 in which at least one of the precursorions has a mass/charge ratio of 433.0±0.5 and generating one or morefragment ions of an angiotensin 1 precursor ion from the groupconsisting of ions with a mass/charge ratio of 619.4±0.5 or 647.4±0.5.In some embodiments, step (b) comprises purification of angiotensin 1from the sample with a solid-phase extraction column. In furtherembodiments, the water stable protease inhibitor isaminoethylbenzylsulfonyl fluoride. In some embodiments, a degradationstandard may be used to determine the degree of degradation ofangiotensin 1 during a renin activity assay.

In another aspect, methods are provided for measuring the renin activityin a sample. The methods include: (a) incubating the sample underconditions suitable for the generation of angiotensin 1 by renin in thesample; (b) purifying angiotensin 1 in the sample by liquidchromatography; (c) ionizing the purified angiotensin 1 to produce oneor more ions detectable by mass spectrometry; (d) detecting the amountof the angiotensin 1 ion(s) by mass spectrometry, wherein the ion(s) areselected from the group consisting of ions with a mass/charge ratio of433.0±0.5, 619.4±0.5, 647.4±0.5 and 1297±0.5; (e) using the amount ofion(s) detected to measure the amount of angiotensin 1 in said sample;and (f) using the amount of angiotensin 1 in the sample to calculaterenin activity in the sample. In some embodiments, the limit ofquantitation of the methods is less than or equal to 0.1 ng/mL; lessthan or equal to 0.05 ng/mL; or about 0.03 ng/mL. In some embodiments,the incubation occurs in the presence of a water stable proteaseinhibitor. In related embodiments, the water stable protease inhibitoris aminoethylbenzylsulfonyl fluoride. In some embodiments, the liquidchromatography is high performance liquid chromatography (HPLC). In someembodiments, step (b) of the method further comprises purification ofangiotensin 1 with a solid-phase extraction column. In furtherembodiments, the amount of the angiotensin 1 ion(s) is related to theamount of angiotensin 1 in the test sample by comparison to an internalstandard. In some embodiments, a degradation standard may be used todetermine the degree of degradation of angiotensin 1 during a reninactivity assay.

In some embodiments, if the PRA is less than 0.65 ng angiotensin per mLper hr after about 3 hours of incubation, the test sample may beincubated for a longer period of time (e.g. up to about 18 hours) toestablish the PRA generation protocol.

Preferred embodiments utilize high performance liquid chromatography(HPLC), alone or in combination with one or more purification methods,for example but not limited to a solid phase extraction technique orprotein precipitation, to purify angiotensin 1 in samples.

In certain embodiments of the methods disclosed herein, massspectrometry is performed in positive ion mode. Alternatively, massspectrometry can be performed in negative ion mode. In particularlypreferred embodiments, angiotensin 1 is measured using both positive andnegative ion mode. In certain preferred embodiments, angiotensin 1 ismeasured using electrospray ionization (ESI) or matrix assisted laserdesorption ionization (MALDI) in either positive or negative mode.

In certain embodiments, the angiotensin 1 ions detectable in a massspectrometer are selected from the group consisting of ions with amass/charge ratio (m/z) of 1297±0.5, 756±0.5, 649±0.5, 647.4±0.5,619.4±0.5, 534±0.5, 506±0.5, 433.0±0.5, 343±0.5, 255±0.5, and 110±0.5.In particularly preferred embodiments, the precursor ions have amass/charge ratio of 433.0±0.5, and the fragment ions have a mass/chargeratio of 619.4±0.5 or 647.4±0.5.

In certain embodiments, a separately detectable isotope-labeledangiotensin 1, is added to the sample as an internal standard. In theseembodiments, all or a portion of both the endogenous angiotensin 1 andthe internal standard present in the sample is ionized to produce aplurality of ions detectable in a mass spectrometer, and one or moreions produced from each are detected by mass spectrometry. In relatedembodiments, the isotope labeled angiotensin 1 may comprise ¹³C and ¹⁵Nisotope labeled valine, arginine, isoleucine, leucine, lysine,phenylalanine proline subunits or combinations thereof. In furtherpreferred embodiments, the isotope labeled angiotensin 1 has the valinesubunit where carbon atoms are substituted with ¹³C isotopes and thenitrogen atom is replaced with ¹⁵N isotopes leading to an increase inmass of 6 Da relative to natural angiotensin 1. In related preferredembodiments, the isotope labeled angiotensin 1 has valine and isoleucinesubunits where carbon atoms are substituted with ¹³C isotopes and thenitrogen atoms are replaced with ¹⁵N isotopes. The mass of this isotopelabeled angiotensin 1 is nominally 13 Da higher than natural angiotensin1.

In preferred embodiments, the presence and/or amount of the angiotensin1 ion(s) is related to the presence and/or amount of angiotensin 1 inthe test sample by comparison to the internal standard.

In certain preferred embodiments of the aspects disclosed herein, thelimit of quantitation (LOQ) of angiotensin 1 is less than or equal to0.1 ng/mL; such as less than or equal to 0.05 ng/mL; such as about 0.03ng/mL; and the upper limit of quantitation (ULOQ) of angiotensin 1 isgreater than or equal to 100,000 fmol/mL.

In another aspect, kits are provided for an angiotensin 1 quantitationassay. The kits comprise aminoethylbenzylsulfonyl fluoride (AEBSF) inphosphate buffered saline solution, wherein said AEBSF in phosphatebuffered saline solution is present in amounts sufficient for at leastone assay. The kits may additionally comprise internal standard andmaleic acid in amounts sufficient for at least one assay.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Samples may be purified herein by various means to allowremoval of one or more interfering substances, e.g., one or moresubstances that would interfere with the detection of selectedangiotensin 1 parent and daughter ions by mass spectrometry.

As used herein, the term “test sample” refers to any sample that maycontain angiotensins. As used herein, the term “body fluid” means anyfluid that can be isolated from the body of an individual. Examples ofbody fluids include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of separationtechniques which employ “liquid chromatography” include reverse phaseliquid chromatography (RPLC), high performance liquid chromatography(HPLC), and turbulent flow liquid chromatography (TFLC) (sometimes knownas high turbulence liquid chromatography (HTLC) or high throughputliquid chromatography). In some embodiments, an SPE column may be usedin combination with an LC column. For example, a sample may be purifiedwith a TFLC extraction column, followed by additional purification witha HPLC analytical column.

As used herein, the term “high performance liquid chromatography” or“HPLC” (sometimes known as “high pressure liquid chromatography”) refersto liquid chromatography in which the degree of separation is increasedby forcing the mobile phase under pressure through a stationary phase,typically a densely packed column.

As used herein, the term “turbulent flow liquid chromatography” or“TFLC” (sometimes known as high turbulence liquid chromatography or highthroughput liquid chromatography) refers to a form of chromatographythat utilizes turbulent flow of the material being assayed through thecolumn packing as the basis for performing the separation. TFLC has beenapplied in the preparation of samples containing two unnamed drugs priorto analysis by mass spectrometry. See, e.g., Zimmer et al., J ChromatogrA 854: 23-35 (1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368,5,795,469, and 5,772,874, which further explain TFLC. Persons ofordinary skill in the art understand “turbulent flow”. When fluid flowsslowly and smoothly, the flow is called “laminar flow”. For example,fluid moving through an HPLC column at low flow rates is laminar. Inlaminar flow the motion of the particles of fluid is orderly withparticles moving generally in straight lines. At faster velocities, theinertia of the water overcomes fluid frictional forces and turbulentflow results. Fluid not in contact with the irregular boundary “outruns”that which is slowed by friction or deflected by an uneven surface. Whena fluid is flowing turbulently, it flows in eddies and whirls (orvortices), with more “drag” than when the flow is laminar. Manyreferences are available for assisting in determining when fluid flow islaminar or turbulent (e.g., Turbulent Flow Analysis: Measurement andPrediction, P. S. Bernard & J. M. Wallace, John Wiley & Sons, Inc.,(2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott,Cambridge University Press (2001)).

As used herein, the term “solid phase extraction” or “SPE” refers to aprocess in which a chemical mixture is separated into components as aresult of the affinity of components dissolved or suspended in asolution (i.e., mobile phase) for a solid through or around which thesolution is passed (i.e., solid phase). In some instances, as the mobilephase passes through or around the solid phase, undesired components ofthe mobile phase may be retained by the solid phase resulting in apurification of the analyte in the mobile phase. In other instances, theanalyte may be retained by the solid phase, allowing undesiredcomponents of the mobile phase to pass through or around the solidphase. In these instances, a second mobile phase is then used to elutethe retained analyte off of the solid phase for further processing oranalysis. SPE, including utilization of a turbulent flow liquidchromatography (TFLC) column as an extraction column, may operate via aunitary or mixed mode mechanism. Mixed mode mechanisms utilize ionexchange and hydrophobic retention in the same column; for example, thesolid phase of a mixed-mode SPE column may exhibit strong anion exchangeand hydrophobic retention; or may exhibit column exhibit strong cationexchange and hydrophobic retention.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. In a preferred embodiment the analytical column containsparticles of about 4 μm in diameter.

As used herein, the term “on-line” or “inline,” for example as used in“on-line automated fashion” or “on-line extraction,” refers to aprocedure performed without the need for operator intervention. Forexample, by careful selection of valves and connector plumbing, thesolid phase extraction and liquid chromatography columns can beconnected as needed such that material is passed from one to the nextwithout the need for any manual steps. In preferred embodiments, theselection of valves and plumbing is controlled by a computerpre-programmed to perform the necessary steps. Most preferably, thechromatography system is also connected in such an on-line fashion tothe detector system, e.g., an MS system. Thus, an operator may place atray of multi-well or multi-tube samples in an autosampler and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected. In contrast, the term“off-line” as used herein refers to a procedure requiring manualintervention of an operator. Thus, if samples are subjected toprecipitation, and the supernatants are then manually loaded into anautosampler, the precipitation and loading steps are off-line from thesubsequent steps. In various embodiments of the methods, one or moresteps may be performed in an on-line automated fashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z.” MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces”; 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry”; 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry”; 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes”; Wright et al., Prostate Cancer and ProstaticDiseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis21:1164-67 (2000).

As used herein, the term “operating in positive ion mode” refers tothose mass spectrometry methods where positive ions are generated anddetected. The term “operating in negative ion mode” as used herein,refers to those mass spectrometry methods where negative ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber, which is heated slightly to prevent condensation and toevaporate solvent. As the droplets get smaller the electrical surfacecharge density increases until such time that the natural repulsionbetween like charges causes ions as well as neutral molecules to bereleased.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectroscopy methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESE foranalyzing less-polar species.

The term “Atmospheric Pressure Photoionization” or “APPI” as used hereinrefers to the form of mass spectroscopy where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. See, e.g., Robb et al., Atmosphericpressure photoionization: An ionization method for liquidchromatography-mass spectrometry. Anal. Chem. 72(15): 3653-59 (2000).

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term in “field desorption” refers to methods inwhich a non-volatile test sample is placed on an ionization surface, andan intense electric field is used to generate analyte ions.

As used herein, the term in “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a precision of 20% and anaccuracy of 80% to 120%. The upper limit of quantitation refers to theupper quantifiable linear range of analyte response.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is defined arbitrarily as 3 standard deviations (SD) fromthe zero concentration.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.5 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows collision-induced dissociation full scan spectra for Ang1(m/z=433.0).

FIG. 2 shows collision-induced dissociation full scan spectra forinternal standard (m/z=434.8).

FIG. 3 shows an exemplary calibration curve for the mass spectrometricdetection of Ang1.

FIGS. 4 A, B, and C show exemplary mass chromatograms of Ang1(m/z=433.0±0.5), internal standard (m/z=434.8±0.5), and degradationstandard (m/z=437.3±0.5), respectively, for a low concentrationcalibrator.

FIGS. 5 A, B, and C show exemplary mass chromatograms of Ang1(m/z=433.0±0.5), internal standard (m/z=434.8±0.5), and degradationstandard (m/z=437.3±0.5), respectively, for a patient sampledemonstrating 0.1 ng/mL/hr.

FIG. 6 shows the fraction of intact degradation standard observed as afunction of the observed PRA in several patient samples.

FIG. 7 shows a total ion chromatogram (with background subtracted)collected over retention times (RT) of 0.00 to about 2.49 minutes forprecursor ion scanning across a m/z range of about 300.00 to about650.00 for a highly degenerative sample.

FIG. 8 shows precursor ion scanning spectra generated at RT of about1.30 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 9 shows precursor ion scanning spectra generated at RT of about1.23 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 10 shows precursor ion scanning spectra generated at RT of about1.17 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 11 shows precursor ion scanning spectra generated at RT of about1.13 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 12 shows precursor ion scanning spectra generated at RT of about0.97 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 13 shows precursor ion scanning spectra generated at RT of about0.89 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

FIG. 14 shows precursor ion scanning spectra generated at RT of about0.68 minutes across a m/z range of about 300.00 to about 650.00 for ahighly degenerative sample.

DETAILED DESCRIPTION OF THE INVENTION

Methods are described for measuring the amount of angiotensin 1 in asample. More specifically, methods are described for detecting andquantifying angiotensin 1 related to renin activity in a plasma sample.The methods utilize liquid chromatography (LC), most preferably HPLC, toperform a purification of selected analytes, and combine thispurification with unique methods of mass spectrometry (MS), therebyproviding a high-throughput assay system for detecting and quantifyingangiotensin 1 in a test sample. The preferred embodiments areparticularly well suited for application in large clinical laboratoriesfor automated PRA assay. The methods provided have enhanced sensitivityand are accomplished in less time and with less sample preparation thanrequired in other PRA assays.

Suitable test samples include any test sample that may contain theanalyte of interest. In some preferred embodiments, a sample is abiological sample; that is, a sample obtained from any biologicalsource, such as an animal, a cell culture, an organ culture, etc. Incertain preferred embodiments, samples are obtained from a mammaliananimal, such as a dog, cat, horse, etc. Particularly preferred mammaliananimals are primates, most preferably male or female humans,Particularly preferred samples include blood, plasma, serum, saliva,cerebrospinal fluid, or other tissue sample. Such samples may beobtained, for example, from a patient; that is, a living person, male orfemale, presenting oneself in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition. The test sample ispreferably obtained from a patient, for example, blood serum or plasma.To avoid irreversible cryoactivation of plasma prorenin, samples shouldbe processed immediately at room temperature or stored completely frozenand quickly thawed just prior to use.

Various incubation conditions may be used to facilitate the preparationand generation of angiotensin 1 by renin prior to chromatography and orMS sample analysis so that the analysis can be automated. The currentinvention incorporates a single addition of a reagent cocktail suitableto perform the generation of angiotensin 1. The reagent cocktail is madefrom pre-mixing all the reagents in one simple step. This singlegeneration of a reagent cocktail is achieved by replacing the typicallyutilized water-sensitive enzyme inhibitor, e.g., phenylmethylsulphonylfluoride (PMSF), with a water stable, e.g., aminoethylbenzylsulfonylfluoride (AEBSF). This is especially useful in automation for a largesample size because the labor intensive assay setup requires mixing ofthe water based reagents and a water-sensitive enzyme inhibitor, e.g.,PMSF. This sequential addition requires vigorous stirring between eachstep to ensure homogeneity in the reagent solution before incubation.Furthermore, PMSF should be prepared fresh because PMSF is unstable inaqueous media. AEBSF has the added benefit of being much less toxic thanPMSF. According to current invention, the reagent cocktail comprisingthe water-stable AEBSF has an unexpected long stability profile comparedto a PMSF stock solution. The AEBSF-containing regent cocktail can bestored at −20° C. for up to approximately 6 months or at roomtemperature for approximately a week.

The present invention contemplates kits for an angiotensin 1quantitation assay. A kit for an angiotensin 1 quantitation assay of thepresent invention may include a kit comprising AEBSF in phosphatebuffered saline solution, in amounts sufficient for at least one assay.Kits contemplated by the present invention may also comprise isotopelabeled internal standard and maleic acid. If inclusion of a degradationstandard is desired in the kits, generation buffer can be included thatcomprises maleic acid, AEBSF and degradation standard. Typically, thekits will also include instructions recorded in a tangible form (e.g.,contained on paper or an electronic medium) for using the packagedsolutions for use in a measurement assay for determining the amount ofangiotensin 1.

The calibration and QC pools were prepared using mock serum consistingof phosphate buffered saline supplemented with bovine serum albumin at45 mg/mL. No source of human or non-human plasma or stripped serum wasidentified that did not contain measurable amounts of angiotensin 1.

Typically, the frozen plasma samples and controls are thawed rapidly toprevent cryoactivtion of prorenin to renin before incubation. The sampleis incubated at 37° C. for up to about 3 h. If the resulting sample doesnot yield a satisfactory PRA measurement, the incubation time can beextended to up to about 18 h. Samples are then frozen (for example,brought to a temperature of about −20° C.) to stop the incubation andstored in this state until analysis. According to current invention,plasma renin may be incubated at 37° C.±1° with endogenous reninsubstrate (angiotensinogen) at pH 5.7 for about 1 to 2 hours in thepresence of converting enzyme, degradation standard (valine andisoleucine isotope labeled angiotensin 1) and angiotensinase inhibitors(EDTA and AEBSF).

After the conclusion of incubation, the samples may be subject toliquid-liquid extraction or solid phase extraction before LCpurification. In the present invention, extraction of angiotensin 1 isadapted utilizing a suitable solid phase extraction column coupled(either on-line or offline) with HPLC. According to preferredembodiments, the method involves adding formic acid to each sample afterincubation and loading samples directly onto the solid-phase extractioncolumn coupled with HPLC-mass spectrometer.

Liquid chromatography (LC) including high-performance liquidchromatography (HPLC) relics on relatively slow, laminar flowtechnology. Traditional HPLC analysis relies on column packing in whichlaminar flow of the sample through the column is the basis forseparation of the analyte of interest from the sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess and may select HPLC instruments and columns that are suitablefor use with angiotensin 1. The chromatographic column typicallyincludes a medium (i.e., a packing material) to facilitate separation ofchemical moieties (i.e., fractionation). The medium may include minuteparticles. The particles include a bonded surface that interacts withthe various chemical moieties to facilitate separation of the chemicalmoieties. One suitable bonded surface is a hydrophobic bonded surfacesuch as an alkyl bonded surface. Alkyl bonded surfaces may include C-4,C-8, C-12, or C-18 bonded alkyl groups, preferably C-8 or C-18 bondedgroups. The chromatographic column includes an inlet port for receivinga sample directly or indirectly from coupled SPE column and an outletport for discharging an effluent that includes the fractionated sample.In one embodiment, the sample (or pre-purified sample) may be applied tothe column at the inlet port, eluted with a solvent or solvent mixture,and discharged at the outlet port. Different solvent modes may beselected for eluting the analyte(s) of interest. For example, liquidchromatography may be performed using a gradient mode, an isocraticmode, or a polytyptic (i.e. mixed) mode. During chromatography, theseparation of materials is effected by variables such as choice ofeluent (also known as a “mobile phase”), elution mode, gradientconditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In another embodiment, the solid-phase extraction (SPE) column may beemployed before HPLC on a hydrophobic column chromatographic system. Incertain preferred embodiments, a column comprising polymeric sorbentsmay be coupled on-line with a HPLC system. In certain preferredembodiments, purification of the sample with a SPE column and HPLC areperfoi led using HPLC Grade Ultra Pure 0.1% formic acid in water and0.1% formic acid in acetonitrile as the mobile phases. Preferably, theSPE column used in these embodiments is capable of recovering more than80% of angiotensin from plasma.

By careful selection of valves and connector plumbing, one or more solidphase extraction and chromatographic columns may be connected as neededsuch that material is passed from one to the next without the need forany manual steps. In preferred embodiments, the selection of valves andplumbing is controlled by a computer pre-programmed to perform thenecessary steps. Most preferably, the chromatography system is alsoconnected in such an on-line fashion to the detector system, e.g., an MSsystem. Thus, an operator may place a tray of samples in an autosampler,and the remaining operations are performed under computer control,resulting in purification and analysis of all samples selected.

Mass spectrometry is performed using a mass spectrometer, which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. In various embodiments, angiotensin 1present in a test sample may be ionized by any method known to theskilled artisan. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

In preferred embodiments, angiotensin 1 may be ionized by electrosprayionization (ESI) or matrix assisted laser desorption ionization (MALDI).In further preferred embodiments, angiotensin 1 is ionized by heatedelectrospray ionization (HESI) in positive mode.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio. Suitable analyzers for determining mass-to-chargeratios include triple quadrupole analyzers, quadrupole analyzers, iontraps analyzers, fourier transform analyzers, orbitrap analyzers andtime-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected using ascanning mode, e.g., highly selectively reaction monitoring (H-SRM),multiple reaction monitoring (MRM) or selected reaction monitoring(SRM), or alternatively, ions may be detected using a selective ionmonitoring mode (SIM). Preferably, the mass-to-charge ratio isdetermined using a triple quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument. A linear series of threequadrupoles are known as a triple quadrupole mass spectrometer. Thefirst (Q1) and third (Q3) quadrupoles act as mass filters, and themiddle (Q2) quadrupole is employed as a collision cell. This collisioncell is an RF only quadrupole (non-mass filtering) using He, Ar, or Ngas (˜10⁻³ Torr, ˜30 eV) to induce collisional dissociation of selectedparent ion(s) from Q1. Subsequent fragments are passed through to Q3where they may be filtered or scanned fully.

One may enhance the selectivity of the MS technique by employing “tandemmass spectrometry,” or “MS/MS.” In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu) The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, molecular standards may be run withthe samples, and a standard curve constructed based on ions generatedfrom those standards. Using such a standard curve, the relativeabundance of a given ion may be converted into an absolute amount of theoriginal molecule. In certain preferred embodiments, an internalstandard is used to generate a standard curve for calculating thequantity of angiotensin 1. Methods of generating and using such standardcurves are well known in the art and one of ordinary skill is capable ofselecting an appropriate internal standard. For example, an isotopelabeled angiotensin 1 may be used as an internal standard; in certainpreferred embodiments the standard is isotope labeled angiotensin 1where the valine subunit has been fully substituted with valine wherethe carbon atoms have been substituted with ¹³C isotopes and thenitrogen atoms have been replaced with ¹⁵N isotopes. Numerous othermethods for relating the amount of an ion to the amount of the originalmolecule will be well known to those of ordinary skill in the art.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In certain embodiments, angiotensin 1 is quantitated using MS/MS asfollows. Samples are subjected to liquid chromatography, preferably asolid phase extraction column followed by HPLC, the flow of liquidsolvent from the chromatographic column enters the heated nebulizerinterface of an MS/MS analyzer and the solvent/analyte mixture isconverted to vapor in the heated tubing of the interface. The analyte(e.g., angiotensin 1), contained in the nebulized solvent, is ionized bythe corona discharge needle of the interface, which applies a largevoltage to the nebulized solvent/analyte mixture. The ions, e.g.precursor ions, pass through the orifice of the instrument and enter thefirst quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters,allowing selection of ions (i.e., “precursor” and “fragment” ions) basedon their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collisioncell, where ions are fragmented. The first quadrupole of the massspectrometer (Q1) selects for molecules with the mass to charge ratiosof angiotensin 1. Precursor ions with the correct mass/charge ratios ofangiotensin 1 are allowed to pass into the collision chamber (Q2), whileunwanted ions with any other mass/charge ratio collide with the sides ofthe quadrupole and are eliminated. Precursor ions entering Q2 collidewith neutral collision gas molecules, for example argon gas molecules,and fragment. This process is called collision activated dissociation(CAD). The fragment ions generated are passed into quadrupole 3 (Q3),where the fragment ions of angiotensin 1 are selected while other ionsare eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode. Using standard methods well known in the art, one of ordinaryskill is capable of identifying one or more fragment ions of aparticular precursor ion of angiotensin 1 that may be used for selectionin quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte(angiotensin 1) of interest. In certain embodiments, the area under thecurves, or amplitude of the peaks, for fragment ion(s) and/or precursorions are measured to determine the amount of angiotensin 1. As describedabove, the relative abundance of a given ion may be converted into anabsolute amount of the original analyte, e.g., angiotensin 1, usingcalibration standard curves based on peaks of one or more ions of aninternal molecular standard, such as isotope labeled angiotensin 1.

These data may be relayed to a computer, which generates plots of ioncount versus time. The areas under the peaks are determined andcalibration curves are constructed by plotting standard concentrationversus peak area ratio of Ang1/internal standard. Using the calibrationcurves, the amount of angiotensin 1 and selected internal standards inthe sample is determined. The rate of angiotensin 1 formation over agiven time, i.e., the amount of angiotensin 1 formed during sampleincubation, can be calculated from these determinations. This rate is anindication of renin activity in the sample.

The basic process of reducing data can be performed manually or with theassistance of computer software. The dilution of plasma and generationtime are taken into account in determining the final PRA value accordingto the following calculation:

${\frac{{x\mspace{14mu} {uL}\mspace{14mu} {plasma}} + {y\mspace{14mu} {uL}\mspace{14mu} {buffer}}}{x\mspace{14mu} {uL}\mspace{14mu} {plasma}}*\frac{1}{z\mspace{14mu} {hours}}} = f$

wherein x is the volume of the plasma sample; y is the volume of bufferadded and z is the time for sample incubation.

The result from LCquan reported as ng/mL is then multiplied by f to givea corrected measurement for the PRA assay in ng per mL per hr. If thesample has been diluted to bring the final result into the linear rangeof the assay, the final result is also multiplied by the degree ofdilution.

Some plasma samples have a high degree of enzymatic activity whichdegrades angiotensin 1 during a renin activity assay. In someembodiments, a degradation standard may be used to determine the degreeof degradation of angiotensin 1 that has occurred during generation ofangiotensin 1 by renin. In these embodiments, the degradation standardis added to the sample prior to incubation. Procedurally, this differsfrom the timing of adding an internal standard, which is added afterincubation but prior to mass spectrometric analysis. In someembodiments, the degree of degradation may be determined by comparisonof the measured quantity of degradation standard (after incubation) withthe measured quantity of internal standard. In other embodiments, thedegree of degradation may be determined by identifying and quantifyingspecies in the sample that are generated by the breakdown of thedegradation standard. Typically, breakdown products useful formonitoring are essentially absent from the sample but for the breakdownof the degradation standard. The degradation standard may be a syntheticpeptide which incorporates one or more ¹³C and/or ¹⁵N labeled aminoacids, that when ionized produces at least one ion with a m/z differentthan ions produced by ionizing unlabeled angiotensin 1 and the internalstandard. Calculation of a degradation factor in such embodimentsrequires further consideration. In embodiments where the measured amountof degradation standard remaining after incubation is compared to themeasured amount of internal standard, the percent degradation may becalculated as follows.

First, the baseline ratio (BR) is calculated by calculating the ratio ofthe average area of degradation standard (DS) to analytical internalstandard (IS) for the three Bio-Rad QC samples:

${\frac{{Low}\mspace{14mu} {DS}}{{Low}\mspace{14mu} {IS}} + \frac{{Med}\mspace{14mu} {DS}}{{Med}\mspace{14mu} {IS}} + {\frac{{High}\mspace{14mu} {DS}}{{High}\mspace{14mu} {IS}} \times \frac{1}{3}}} = {{baseline}\mspace{14mu} {ratio}\mspace{14mu} ({BR})}$

Then, the % degradation for each patient sample is calculated:

${\frac{DS}{IS} \times \frac{1}{BR} \times 100} = {\% \mspace{14mu} {degradation}}$

In embodiments where generation of breakdown products of the degradationstandard are quantitated, MRM transitions to monitor with tandem massspectrometry may be identified by scanning for precursor ions associatedwith N- or C-terminal directed degradation of the degradation standard.Degradation standards may be selected such that these precursor ions maybe identified by their characteristic fragmentation into known fragmentions. For example, in some embodiments, the degradation standard maycomprise isotope-labeled Ang1 peptides. Preferred isotope-labeled Ang1peptides include those containing one or more modified amino acidresidues selected from the group consisting of modified valine,isoleucine, proline, or histidine. In embodiments where the degradationstandard comprises isotope-labeled Ang1 peptides containing modifiedproline or histidine, Q3 may be set to select for immonium ions ofisotope-labeled proline or histidine, while Q1 is scanned over m/zranges that would be associated with N- or C-terminal degradation of thelabeled Ang1 peptides. Quantitation may be accomplished by use of totalion current resulting from such precursor ion scans.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample and Reagent Preparation

Reagents: angiotensin 1, aminoethylbenzylsulfonyl fluoride (AEBSF),bovine serum albumin (protease free), maleic anhydride, phosphatebuffered saline tablets (PBS tablets), formic acid, and Bio-RadLyphocheck controls were purchased from their respective suppliers.Isotope labeled Ang1 standards were custom synthesized. The internalstandard was the single isotope labeled Ang1 where natural valine iswholly substituted with valine containing ¹³C and ¹⁵N atoms leading to amass difference of +6 Da. Similarly, the degradation standard used inthe assay was double isotope labeled Ang1 where natural valine andisoleucine are wholly substituted with valine and isoleucine containing¹³C and ¹⁵N atoms leading to a mass difference of +13 Da.

The analytical internal standard (including degradation standard) may bea synthetic peptide which incorporates one or more ¹³C and/or ¹⁵Nlabeled amino acids. The primary sequence of the internal standard usedin this example is valine-isotope labeled Ang1, DR[V¹⁵N, ¹³C]YIHPFHL.Synthesis was accomplished at the 2-5 mg scale with a final purityspecification of >95%. The peptide was packaged in individual aliquotswith a total peptide content of 2 nmol per vial. In addition, adegradation standard with valine and isoleucine-isotope labeled Ang1,i.e. DR[V¹⁵N, ¹³C]Y[I¹⁵N, ¹³C]HPFHL, was prepared. Synthesis wasaccomplished at the 2-5 mg scale with a final purity specificationof >95%. The peptide was packaged in individual aliquots with a totalpeptide content of 2 nmol per vial.

Doubly distilled deionized water and HPLC-grade methanol andacetonitrile were used throughout the investigation. Mock serum wereprepared according to the following procedure. First, 300 mL of waterwas added to a graduated cylinder along with two PBS tablets, Then, 22.5g of bovine serum albumin was added and thoroughly mixed. Next, 0.02 gAEBSF was added and thoroughly mixed. Finally, sufficient water wasadded to reach a final volume of 500 mL.

Preparation of generation cocktail, i.e., incubation solution, isdependent on the number of samples in a given assay setup. The volumesindicated in Table 1 (below) include a 20% excess to assure sufficientvolume for transfer. Maleic acid, AEBSF, and degradation standard (e.g.DR[V¹⁵N, ¹³C]Y[I¹⁵N, ¹³C]HPFHL) were mixed in a 15 mL PP tube.

TABLE 1 Volumes useful for preparing generation cocktails. No. ofsamples Maleic acid AEBSF stock Degradation standard 150 4.75 mL 250 uL22 uL 225 7.13 mL 375 uL 33 uL 300  9.5 mL 500 uL 44 uL

AEBSF stock solution was evaluated for its stability. 0.91 gams of AEBSFpowder was placed into a 15 mL Corning tube and dissolved with 10 mL ofpeptide solvent. 0.5 mL aliquots of this solution were placed into 1.5mL Nunc cyrovials. Stored in this way, the solution was found to bestable for up to about 6 months when stored at −20° C., or for about oneweek when stored at room temperature.

Example 2 Calibrators and Controls

Calibrators was prepared by spiking known amounts of angiotensin 1 intomatrix using mock serum. The concentration of the calibration series was0.0 (blank), 0.14, 0.27, 1.1, 2.19, 8.79, 35.15, and 43.2 ng/mL. QCpools were prepared at 1.1, 8.7, and 35 ng/mL. Controls were obtainedfrom BioRad and consisted of samples with high, intermediate, and lowrenin activity. QC pools and calibrators were ran with each sample batchfor quality assurance.

Example 3 Preparation of Samples for Generation

The frozen samples were quickly brought to room temperature using a fanor room temperature water bath. The specimens were intermittentlyinverted to hasten the thawing process as quick thawing is critical toprevent the cryoactivation of prorenin to renin. The samples wereallowed to remain at room temperature once thawed. All plasma sampleswere vortexed thoroughly prior to testing.

Example 4 Incubation Procedure

250 uL of EDTA plasma was mixed with 25 uL of generation cocktail (0.275M Maleic acid, 1 mM AEBSF, 4 uM degradation standard). Samples wereincubated at about 37 degrees for about 1-3 hours and subsequently mixedwith an approximately equal volume of 10% formic acid containing 2 uManalytical internal standard.

Example 5 Purification of Angiotensin I from Test Sample

All chromatographic steps were carried out using a Cohesive TLX fourchannel system.

100 uL of acidified sample was injected onto a 2.1 mm×20 mm 25 uM HLBextraction cartridge using a solvent A (0.1% Formic acid in water). Theextraction cartridge was eluted using 100 uL of and 80:20 mixture ofsolvent A (0.1% Formic acid in water) and solvent B (0.1% Formic acid inacetonitrile). The eluate was mixed at a ratio of 1:3 with solvent A anddirected to a 2.1 mm×50 mm 5 uM Xbridge 130 BEH analytical column. Aftertransfer of the analyte to the analytical column, a gradient from 95% Ato 65% solvent A was developed, At the appropriate time during thegradient, the flow was diverted from waste and directed to the massspectrometer. Both the extraction cartridge and analytical column werecleaned and re-equilibrated in-situ before the next injection.

Example 6 Detection and Quantitation of Angiotensin 1 by MS/MS

MS/MS was performed using a TSQ Quantum Ultra with a HESI (HeatedElectrospray Ionization) source in positive ion mode. Selected reactionmonitoring was used for quantitative analysis of the analyte andinternal standards. Six transitions were monitored, two for each ofanalyte, analytical internal standard, and degradation standard. Peakarea ratios between analyte and internal standard were calibratedagainst a series of known angiotensin 1 stocks and calibration curveswere then constructed using a 1/× weighted linear regression. SelectedMS/MS parameters are listed in Table 2, below:

TABLE 2 Selected MS/MS parameters. Ionization voltage 3000 V VaporizerTemperature 300° C. Sheath Gas 30 units Ion Sweep Gas Pressure  0Auxiliary Gas Pressure 30 units Capillary Temperature 350° C. Tube LensOffset 110 Skimmer Offset 0 to −5 V Collision Pressure 1.5 mTorrCollision Energy 16 V (433→ 619) 22 V (433→ 647)

Ions passed to the first quadrupole (Q1), which was set to select ionswith a mass to charge ratio of 433.0±0.5 m/z [M+3H]³⁺. Ions enteringquadrupole 2 (Q2) collided with argon gas to generate ion fragments,which were passed to quadrupole 3 (Q3) for further selection.Simultaneously, the same process using isotope dilution massspectrometry was carried out with an isotope labeled angiotensin 1internal standard or a isotope labeled angiotensin 1 degradationstandard. The following mass transitions were used for detection andquantitation during validation on positive polarity.

TABLE 3 Mass Transitions for angiotensin 1 (Positive Polarity) PrecursorIon Product Ions Analyte (m/z) (m/z) Angiotensin I 433.0 ± 0.5 647.4 ±0.5, [M + 3H]³⁺ 619.4 ± 0.5 Internal Standard 434.8 ± 0.5 653.4 ± 0.5,(DR[V¹⁵N, ¹³C]YIHPFHL) [M + 3H]³⁺ 625.5 ± 0.5 Degradation Standard 437.3± 0.5 660.4 ± 0.5, (DR[V¹⁵N, ¹³C]Y[I¹⁵N, ¹³C]HPFHL) [M + 3H]³⁺ 631.4 ±0.5

These data were relayed to a computer, which generated plots of ioncount versus time. The areas under the peaks were determined andcalibration curves were constructed by plotting standard concentrationversus peak area ratio of analyte/internal standard. Using thecalibration curves, the concentrations of angiotensin 1 and selectedinternal standards were quantitated for samples. An exemplarycalibration curve is shown in FIG. 3. Exemplary mass chromatograms ofAng1 (m/z=433.0±0.5), internal standard (m/z=434.8±0.5), and degradationstandard (m/z=437.3±0.5), respectively, for a low concentrationcalibrator are shown in FIGS. 4 A, B, and C. Similarly, FIGS. 5 A, B,and C show exemplary mass chromatograms of Ang1 (m/z=433.0±0.5),internal standard (m/z=434.8±0.5), and degradation standard(m/z=437.3±0.5), respectively, for a patient sample demonstrating 0.1ng/mL/hr PRA.

Example 7 Intra-Assay and Inter-Assay Precision and Accuracy

Eight aliquots from each of the three QC pools were analyzed in a singleassay to determine the reproducibility (CV) of a sample within an assay.The following values were determined:

TABLE 4 Inter- and Intra-Assay Variation Low QC Med. QC High QC (1.1ng/mL) (8.7 ng/mL) (35 ng/mL) Inter-assay 6.04% 6.96% 5.64% Intra-assay6.18% 5.14% 5.09%

Example 8 Analytical Sensitivity: Limit of Detection (LOD) and Limit ofQuantitation (LOQ)

The limit of detection (LOD) is the point at which a measured value islarger than its associated uncertainty. The angiotensin 1 zero standardwas run in 17 replicates and the resulting area ratios were backcalculated to a concentration based on the calibrators to determine thelimit of detection of the assay. The limit of detection (LOD) for theangiotensin 1 assay was 30 fmol/m L.

To determine the limit of quantitation (LOQ) with a precision of 20% andan accuracy of 80% to 120%, five different samples at concentrationsclose to the expected LOQ were assayed and the reproducibilitydetermined for each. The LOQ for the angiotensin 1 assay was defined at0.03 ng/mL

Example 9 Assay Reportable Range and Linearity

To establish the linearity of angiotensin 1 detection in the assay, oneblank assigned as zero standard and eight spiked artificial serumsamples (calibrators) were prepared and analyzed on five separate days.A weighted (1/x) linear regression from five consecutive runs yieldedcoefficient correlation of 0.995 or greater, with an accuracy of ±20%revealing a quantifiable linear range of 77 to 100,000 fmol/mL.

Example 10 Plasma Renin Activity Calculations for 3 Hours Incubation

The basic process of reducing data was performed for 3 hours incubation.The dilution of plasma and generation time were taken into account indetermining the final PRA value for samples analyzed in Examples 3-9,above, according to the following calculation:

${\frac{{250\mspace{14mu} {uL}\mspace{14mu} {plasma}} + {25\mspace{14mu} {uL}\mspace{14mu} {buffer}}}{250\mspace{14mu} {uL}\mspace{14mu} {plasma}}*\frac{1}{3\mspace{14mu} {hours}}} = 0.367$

The result from LCquan reported as ng/mL was then multiplied by 0.367 togive a corrected measurement for the PRA assay in ng per mL per hr.Though in this particular Example an additional correction was notnecessary, if the sample had been diluted to bring the final result intothe linear range of the assay, the final result would also have beenmultiplied by the degree of dilution.

FIG. 6 shows the fraction of intact degradation standard observed as afunction of the observed PRA in several patient samples.

Example 11 Quantitation of Breakdown Products from Degradation Standard

As an alternative means to assess the degree of degradation of Ang1,generation of products from the breakdown of the degradation standardwere measured. To do so, precursor ion scanning experiments utilized.The experiments were conducted by using isotope-labeled Ang1 peptidescontaining modified proline or histidine as the degradation standard. Tomonitor the accumulation of the degradation products, Q3 was set toselect for immonium ions of isotope-labeled proline or histidine, whileQ1 is scanned over m/z ranges that would be associated with N- orC-terminal degradation of the labeled Ang1 peptides. Table 5 contains alist of the m/z ratios of several of the breakdown product precursorions that may be monitored to quantify accumulation of breakdownproducts. In the peptide sequences indicated in the table,isotopically-labeled histidines are indicated by “J”.

FIG. 7 shows a total ion chromatogram collected over retention times(RT) of 0.00 to about 2.49 minutes for precursor ion scanning over a m/zrange of about 300.00 to about 650.00. FIGS. 8-14 show spectra generatedfrom precursor ion scanning over a m/z range of about 300.00 to about650.00 at various retention times. These spectra show the relativeabundance of several degradation standard breakdown product precursorions indicated in Table 5.

For example, FIG. 8 (generated at RT of approximately 1.30 minutes)demonstrates m/z peaks at about 344.9±0.5 and about 516.0±0.5 whichcorrespond to the breakdown product VYIJPFJL.

FIG. 9 (generated at RT of approximately 1.23 minutes) demonstrates m/zpeaks at about 311.7±0.5 and about 466.8±0.5 which correspond to thebreakdown product YIJPFJL.

FIG. 10 (generated at RT of approximately 1.17 minutes) demonstrates m/zpeaks at about 350.8±0.5 and about 525.4±0.5 which correspond to thebreakdown product DRVYIJPF, and m/z peaks at about 396.9±0.5 and about594.8±0.5 which correspond to the breakdown product RVYIJPFJL.

FIG. 11 (generated at RT of approximately 1.13 minutes) demonstrates m/zpeaks at about 350.7±0.5 and about 525.2±0.5 which correspond to thebreakdown product DRVYIJPF, a m/z peak at about 385.4±0.5 whichcorrespond to the breakdown product IJPFJL, and m/z peaks at about396.8±0.5 and about 594.9±0.5 which correspond to the breakdown productDRVYIJPFJ.

FIG. 12 (generated at RT of approximately 0.97 minutes) demonstrates m/zpeaks at about 397.4±0.5 and about 595.6±0.5 which correspond to thebreakdown product DRVYIJPFJ.

FIG. 13 (generated at RT of approximately 0.89 minutes) demonstrates am/z peak at about 328.9±0.5 which corresponds to the breakdown productJPFJL.

FIG. 14 (generated at RT of approximately 0.68 minutes) demonstrates am/z peak at about 451.9±0.5 which corresponds to the breakdown productDRVYIJP.

TABLE 5 M/Z ratios for various breakdown products of the degradationstandard Degradation Standard Breakdown Product Precursor Ions (m/z± 0.5) Peptide 1+ 2+ 3+ 4+ DRVYIJPFJL 1302.7 651.8 434.9 326.4 DRVYIJPFJ1189.6 595.3 397.2 RVYIJPFJL 1187.7 594.3 396.5 RVYIJPFJ 537.8 358.9DRVYIJPF 1049.6 525.2 350.5 VYIJPFJL 1031.6 516.0 344.5 RVYIJPF 467.8312.2 YIJPFJL 932.5 466.7 311.5 VYIJPFJ 459.8 306.8 DRVYIJP 902.5 451.7301.5 DRVYIJ 805.4 403.2 RVYIJP 394.2 VYIJPF 389.7 IJPFJL 769.4 385.2JPFJL 656.4 328.7 PFJL 516.3

Example 12 Identification of Patient Sub-population with HighDegradation

A number of samples analyzed for the PRA according to methods outlinedin the above Examples exhibited substantial loss (more than about 40%loss) of the degradation standard during incubation. If fact, asignificant number of patient samples exhibited complete loss of thedegradation standard during degradation. Experiments were conducted onseveral of these samples (n=371) to define a subpopulation of patientswhere observed low PRA is actually due to high levels of Ang1degradation during incubation, rather than low renin activity.

The samples selected for analysis were specifically selected for furtherstudy based on previous PRA values determined by RIA with the intentionof sampling a large number of patients with low PRA values (>0.65ng/mL/hr). Patient samples were separated into 4 categories based on PRAvalues determined by RIA and LC-MS/MS: Group 1 with PRA values between<0.1-0.4 ng/m/hr (n=63); Group 2 with PRA values between 0.4-0.7 ng/m/hr(n=50); Group 3 with PRA values between 0.7-3.0 ng/m/hr (n=108); andGroup 4 with PRA values of >3.0 ng/m/hr (150).

Of the samples in Group 1, eight samples exhibited a complete loss ofdegradation standard after three hours of incubation. Thus, the very lowPRA values measured in about 12% (8 out of 63) of samples in Group 1 mayresult from high Ang1 degradation, rather than from a true lack of reninactivity. However, the overall frequency of these highly degradingsamples in the entire sample population is low (approximately 2%).

FIG. 14 shows the fraction degradation standard recovered intact as afunction of PRA for

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for measuring the amount of angiotensin 1 in a sample, saidmethod comprising: (a) ionizing angiotensin 1 from said sample toproduce one or more ions detectable by mass spectrometry; (b) detectingthe amount of the angiotensin ion(s) by mass spectrometry, said ionsselected from the group consisting of ions with a mass/charge ratio of433.0±0.5, 619.5±0.5, 647.4±0.5 and 1297±0.5; and (c) using the amountof ion(s) detected to measure the amount of angiotensin 1 in saidsample.
 2. The method of claim 1, wherein said method has a limit ofquantitation less than or equal to 0.1 ng/mL.
 3. The method of claim 1,wherein said method further comprises purifying angiotensin 1 from saidsample by high performance liquid chromatography (HPLC).
 4. The methodof claim 1, wherein said method further comprises purifying angiotensin1 from said sample with a solid-phase extraction column.
 5. The methodof claim 1, wherein the amount of the angiotensin 1 ion is related tothe presence or amount of angiotensin 1 in the test sample by comparisonto an internal standard.
 6. A method for measuring the amount ofangiotensin 1 in a sample, said method comprising: (a) incubating saidsample premixed with water stable protease inhibitor that is noteffective against renin under conditions suitable for the generation ofangiotensin 1 by renin in the sample; (b) purifying angiotensin 1 insaid sample by liquid chromatography; (c) ionizing the purifiedangiotensin 1 from said sample to produce one or more ions detectable bymass spectrometry; (d) detecting the amount of the angiotensin 1 ion(s)by mass spectrometry; and (e) using the amount of ion(s) detected tomeasure the amount of angiotensin 1 in said sample.
 7. The method ofclaim 6, wherein said method has a limit of quantitation less than orequal to 0.1 ng/mL.
 8. The method of claim 6, wherein said angiotensin 1ions comprise one or more ions selected from the group consisting ofions with a mass/charge ratio of 433.0±0.5, 619.5±0.5, 647.4±0.5 and1297±0.5.
 9. The method of claim 6, wherein said ionizing comprisesgenerating a precursor ion with a mass/charge ratio of 433.0±0.5, andgenerating one or more fragment ions selected from the group consistingof ions with a mass/charge ratio of 619.5±0.5 and 647.4±0.5.
 10. Themethod of claim 6, wherein the amount of the angiotensin 1 ion isrelated to the presence or amount of angiotensin 1 in the test sample bycomparison to an internal standard.
 11. The method of claim 6, whereinsaid step (b) comprises purifying angiotensin 1 from said sample with asolid-phase extraction column.
 12. The method of claim 6, wherein saidwater stable protease inhibitor is aminoethylbenzylsulfonyl fluoride.13. The method of claim 6, further comprising measuring the degree ofdegradation of angiotensin 1 in the sample by adding a degradationstandard to the sample prior to incubation and measuring the amount ofdegradation standard remaining after incubation.
 14. The method of claim6, further comprising measuring the degree of degradation of angiotensin1 in the sample by adding a degradation standard to the sample prior toincubation and measuring the amount of breakdown products fromdegradation of the degradation standard in the sample after incubation.15. A method for measuring the amount of angiotensin 1 generated byrenin in a sample, said method comprising: (a) incubating the sampleunder conditions suitable for the generation of angiotensin 1 by reninin the sample; (b) purifying angiotensin 1 in said sample by solid phaseextraction; (c) further purifying angiotensin 1 in said sample by liquidchromatography with on-line processing; (d) ionizing the purifiedangiotensin 1 from said sample to produce one or more ions detectable bymass spectrometry; (e) detecting the amount of the angiotensin 1 ion(s)by mass spectrometry, (f) using the amount of ion(s) detected to measurethe amount of angiotensin 1 in said sample.
 16. The method of claim 15,wherein said liquid chromatography is high performance liquidchromatography (HPLC).
 17. The method of claim 15, wherein said methodhas a limit of quantitation less than or equal to 0.1 ng/mL.
 18. Themethod of claim 15, wherein said angiotensin 1 ions comprise one or moreions selected from the group consisting of ions with a mass/charge ratioof 433.0±0.5, 619.5±0.5, 647.4±0.5 and 1297±0.5.
 19. The method of claim15, wherein said ionizing comprises generating a precursor ion with amass/charge ratio of 433.0±0.5, and generating one or more fragment ionsselected from the group consisting of ions with a mass/charge ratio of619.5±0.5 or 647.4±0.5.
 20. The method of claim 15, wherein saidincubation in step (a) comprises incubation in the presence of a waterstable protease inhibitor that is not effective against renin.
 21. Themethod of claim 20, wherein said water stable protease inhibitor isaminoethylbenzylsulfonyl fluoride.
 22. The method of claim 15, whereinthe presence or amount of the angiotensin 1 ion is related to thepresence or amount of angiotensin 1 in the test sample by comparison toan internal standard.
 23. The method of claim 15, further comprisingmeasuring the degree of degradation of angiotensin 1 in the sample byincluding a degradation standard in the sample during incubation andmeasuring the amount of degradation standard remaining after incubation.24. The method of claim 15, further comprising measuring the degree ofdegradation of angiotensin 1 in the sample by including a degradationstandard to the sample during incubation and measuring the amount ofbreakdown products from degradation of the degradation standard in thesample after incubation.
 25. A method for measuring renin activity in asample, said method comprising: (a) incubating the sample underconditions suitable for the generation of angiotensin 1 by renin in thesample; (b) purifying angiotensin 1 from said sample by solid phaseextraction; (c) further purifying angiotensin 1 by liquid chromatographywith on-line processing: (d) ionizing the purified angiotensin 1 fromsaid sample to produce one or more ions detectable by mass spectrometry;(e) detecting the amount of the angiotensin 1 ion(s) by massspectrometry, (f) using the amount of ion(s) detected to measure theamount of angiotensin 1 in said sample; and (g) using the amount ofangiotensin 1 measured in said sample to calculate renin activity in thesample.
 26. The method of claim 25, wherein said liquid chromatographyis high performance liquid chromatography (HPLC).
 27. The method ofclaim 25, wherein said method has a limit of quantitation less than orequal to 0.1 ng/mL.
 28. The method of claim 25, wherein said angiotensin1 ions comprise one or more ions selected from the group consisting ofions with a mass/charge ratio of 433.0±0.5, 619.5±0.5, 647.4±0.5 and1297±0.5.
 29. The method of claim 25, wherein said ionizing comprisesgenerating a precursor ion with a mass/charge ratio of 433.0±0.5, andgenerating one or more fragment ions selected from the group consistingof ions with a mass/charge ratio of 619.5±0.5 or 647.4±0.5.
 30. Themethod of claim 25, wherein said incubation in step (a) comprisesincubation in the presence of a water stable protease inhibitor that isnot effective against renin.
 31. The method of claim 30, wherein saidwater stable protease inhibitor is aminoethylbenzylsulfonyl fluoride.32. The method of claim 25, wherein the presence or amount of theangiotensin 1 ion is related to the presence or amount of angiotensin 1in the test sample by comparison to an internal standard.
 33. The methodof claim 25, further comprising measuring the degree of degradation ofangiotensin 1 in the sample by adding a degradation standard to thesample prior to incubation and measuring the amount of degradationstandard remaining after incubation.
 34. The method of claim 25, furthercomprising measuring the degree of degradation of angiotensin 1 in thesample by adding a degradation standard to the sample prior toincubation and measuring the amount of breakdown products fromdegradation of the degradation standard in the sample after incubation.35. A method for measuring renin activity in a sample, said methodcomprising, (a) incubating the sample premixed with a water stableprotease inhibitor that is not effective against renin under conditionssuitable for the generation of angiotensin 1 by renin in the sample; (b)purifying angiotensin 1 by liquid chromatography; (c) ionizing thepurified angiotensin 1 from said sample to produce one or more ionsdetectable by mass spectrometry; (d) detecting the amount of theangiotensin 1 ion(s) by mass spectrometry; (e) using the amount ofion(s) detected to measure the amount of angiotensin 1 in said sample;and (f) using the amount of angiotensin 1 measured in said sample tocalculate renin activity in the sample.
 36. The method of claim 35,wherein said liquid chromatography is high performance liquidchromatography (HPLC).
 37. The method of claim 35, wherein said methodhas a limit of quantitation less than or equal to 0.1 ng/mL.
 38. Themethod of claim 35, wherein said angiotensin 1 ions comprise one or moreions selected from the group consisting of ions with a mass/charge ratioof 433.0±0.5, 619.5±0.5, 647.4±0.5 and 1297±0.5.
 39. The method of claim35, wherein said ionizing comprises generating a precursor ion with amass/charge ratio of 433.0±0.5, and generating one or more fragment ionsselected from the group consisting of ions with a mass/charge ratio of619.5±0.5 or 647.4±0.5.
 40. The method of claim 35, wherein saidpurifying angiotensin 1 in step (b) comprises purifying angiotensin 1with a solid-phase extraction column.
 41. The method of claim 35,wherein said water stable protease inhibitor is aminoethylbenzylsulfonylfluoride.
 42. The method of claim 35, wherein the presence or amount ofthe angiotensin 1 ion is related to the presence or amount ofangiotensin 1 in the test sample by comparison to an internal standard.43. The method of claim 35, further comprising measuring the degree ofdegradation of angiotensin 1 in the sample by adding a degradationstandard to the sample prior to incubation and measuring the amount ofdegradation standard remaining after incubation.
 44. The method of claim35, further comprising measuring the degree of degradation ofangiotensin 1 in the sample by adding a degradation standard to thesample prior to incubation and measuring the amount of breakdownproducts from degradation of the degradation standard in the sampleafter incubation.
 45. A method for measuring renin activity in a sample,said method comprising, (a) incubating the sample under conditionssuitable for the generation of angiotensin 1 by renin in the sample; (b)purifying angiotensin 1 in said sample by liquid chromatography; (c)ionizing the purified angiotensin 1 from said sample to produce one ormore ions detectable by mass spectrometry; (d) detecting the amount ofthe angiotensin 1 ion(s) by mass spectrometry, said ions selected fromthe group consisting of ions with a mass/charge ratio of 433.0±0.5,619.5±0.5, 647.4±0.5 and 1297±0.5; and (e) using the amount of ion(s)detected to measure the amount of angiotensin 1 in said sample; and (f)using the amount of angiotensin 1 measured in said sample to calculaterenin activity in the sample.
 46. The method of claim 45, wherein saidionizing comprises generating a precursor ion with a mass/charge ratioof 433.0±0.5, and generating one or more fragment ions selected from thegroup consisting of ions with a mass/charge ratio of 619.5±0.5 or647.4±0.5.
 47. The method of claim 45, wherein said method has a limitof quantitation less than or equal to 0.1 ng/mL.
 48. The method of claim45, wherein incubating in step (a) comprises incubating in the presenceof a water stable protease inhibitor that is not effective againstrenin.
 49. The method of claim 45, wherein said water stable proteaseinhibitor is aminoethylbenzylsulfonyl fluoride.
 50. The method of claim45, wherein said purified angiotensin 1 is purified by high performanceliquid chromatography (HPLC).
 51. The method of claim 45, wherein saidmethod further comprises purifying angiotensin 1 from said sample with asolid-phase extraction column.
 52. The method of claim 45, wherein thepresence or amount of the angiotensin 1 ion is related to the presenceor amount of angiotensin 1 in the test sample by comparison to aninternal standard.
 53. The method of claim 45, further comprisingmeasuring the degree of degradation of angiotensin 1 in the sample byadding a degradation standard to the sample prior to incubation andmeasuring the amount of degradation standard remaining after incubation.54. The method of claim 45, further comprising measuring the degree ofdegradation of angiotensin 1 in the sample by adding a degradationstandard to the sample prior to incubation and measuring the amount ofbreakdown products from degradation of the degradation standard in thesample after incubation.
 55. A method for determining whether a patientexhibiting low plasma renin activity levels as determined by the methodof claim 1 is a member of a subpopulation with high natural degradationof angiotensin 1, said method comprising: measuring the degree ofdegradation of angiotensin 1 in the sample by adding a degradationstandard to the sample prior to incubation and measuring the amount ofdegradation standard remaining after incubation.